Engelbert Weis

Quantum efficiency of Photosystem II in relation to 'energy'-dependent quenching of chlorophyll fluorescence *

The balance between light-dependent reactions and electron-consuming reactions in intact sunflower leaves was varied by changing the incident light-flux at constant intercellular CO2 concentration. Measurements of fluorescence quenching were compared to measurements of the rate and apparent quantum yield of whole-chain electron transport at a number of steady-state conditions. The steady-state quantum yield declined with increasing light intensity, falling at the highest intensity to approx. 40% of the maximum value observed in low light. The coefficient for photochemical quenching, qQ, was near 1 in low light and only declined to 0.7 at the highest light, indicating that there was very little feedback from accumulation of reduced electron carriers. On the other hand, there was a large increase in qE, the coefficient for ‘energy’-dependent quenching, as the quantum yield fell. We found that these changes in the steady-state quantum yield, Φs, could be related to the changes in fluorescence quenching by an empirical equation, Φs = qQ(0.32 − 0.17 qE) which accounted for variation in Φs resulting from light saturation or changes in CO2 concentration. We develop a hypothesis that Photosystem (PS) II centers may be converted to an altered state (possibly mediated by the chloroplast ΔpH) which has very little variable fluorescence and a lowered photochemical yield. We develop a kinetic explanation for the properties of the altered form of PS II, and we propose that this mechanism (indicated by qE) functions together with the accumulation of reduced QA (indicated by qQ) to regulate the rate of net photochemistry by PS II when — with increasing light or decreasing CO2 — the potential rate of net photochemistry exceeds that for carbon metabolism. The latter mechanism apparently permits down-regulation of PS II to occur without strong accumulation of reduced QA, except during transients or under the most extreme conditions.

Assimilation images of leaves of Glechoma hederacea: Analysis of non-synchronous stomata related oscillations

An experimental system is described to analyze the spacial distribution of leaf assimilation from chlorophyll-a fluorescence images. Fluorescence parameters were determined with a digitized camera system pixel-by-pixel and calibrated against the assimilation rate from integrated gas exchange. The approach is an extension of the work by P.F. Daley, et al. (1989, Plant Physiol. 90, 1233–1238) and allows the visualisation of assimilation images of leaves with high resolution. With a leaf of Glechoma hederacea L., assimilation images were taken at short time intervals during stomata-related oscillations of the assimilation rate (period about 13 min) over a time period of more than 3 h. Mathematical pixel-by-pixel analysis of images shows nearly Gaussian distribution of assimilation values around a minimum (5 μmol CO2 · m−2 · s−1) and maximum (13 μmol CO2 · m−2 · s−1) peak value during oscillations. Synchronous oscillations, initiated by rapid changes in gas composition, changed into non-synchronous oscillations, due to slight local variations in the period. As a consequence, the oscillation of gas exchange (H2O and CO2) died out whilst leaf assimilation continued to oscillate non-synchronously and patchily. Patchy distribution of frequencies largely followed gas-exchange compartmentation (‘alveoli’), whilst non-homogeneous distribution of the amplitude was on a larger scale, with a decrease in amplitudes from the leaf base towards distal areas. The data are discussed considering the framework of physiological processes involved in stomatal control.

RNA Interference-Mediated Repression of Cell Wall Invertase Impairs Defense in Source Leaves of Tobacco

The significance of cell wall invertase (cwINV) for plant defense was investigated by comparing wild-type tobacco (Nicotiana tabacum) Samsun NN (SNN) with plants with RNA interference (RNAi)-mediated repression of cwINV (SNN∷cwINV). In source leaves of SNN∷cwINV, the activity of cwINV was repressed by about 90%. Sucrose export and apoplastic carbohydrate levels were significantly reduced, while photosynthesis and dark respiration exhibited little or no change. Activities of sucrose synthase and phosphofructokinase were depressed moderately, while ADP-glucose pyrophosphorylase was diminished greatly. Yet, the content of cytosolic/vacuolar carbohydrates was not significantly lower, which correlated with the absence of phenotypic effects in SNN∷cwINV under normal growing conditions. By contrast, defense-related processes in primary metabolism and hypersensitive cell death were impaired and delayed in correlation with repression of cwINV. The increase in cwINV observed in source leaves of the resistant wild type following infection with Phytophthora nicotianae was absent in SNN∷cwINV. Also, defense-related callose deposition at cell-to-cell interfaces, the related decline in sugar export, and accumulation of apoplastic carbohydrates were reduced and delayed. Expression of pathogenesis-related proteins and increase in phenylalanine ammonia-lyase and glucose-6-phosphate dehydrogenase activities were alleviated. Formation of hydrogen peroxide and development of hypersensitive lesions were weak and heterogeneous, and the pathogen was able to sporulate. We conclude that in photosynthetically active leaves of the apoplastic phloem loader, tobacco cwINV plays an essential role for acquisition of carbohydrates during plant-pathogen interactions and that the availability of these carbohydrates supports the onset of the hypersensitive reaction and ensures successful defense.

The Role of Plastocyanin in the Adjustment of the Photosynthetic Electron Transport to the Carbon Metabolism in Tobacco

We investigated adaptive responses of the photosynthetic electron transport to a decline in the carbon assimilation capacity. Leaves of different ages from wild-type tobacco (Nicotiana tabacum) L. var Samsun NN and young mature leaves of tobacco transformants with impaired photoassimilate export were used. The assimilation rate decreased from 280 in young mature wild-type leaves to below 50 mmol electrons mol chlorophyll−1 s−1 in older wild-type leaves or in transformants. The electron transport capacity, measured in thylakoids isolated from the different leaves, closely matched the leaf assimilation rate. The numbers of cytochrome (cyt)-bf complexes and plastocyanin (PC) decreased with the electron transport and assimilation capacity, while the numbers of photosystem I (PSI), photosystem II, and plastoquinone remained constant. The PC to PSI ratio decreased from five in leaves with high assimilation rates, to values below one in leaves with low assimilation rates, and the PC versus flux correlation was strictly proportional. Redox kinetics of cyt-f, PC, and P700 suggest that in leaves with low electron fluxes, PC is out of the equilibrium with P700 and cyt-f and the cyt-f reoxidation rate is restricted. It is concluded that the electron flux is sensitive to variations in the number of PC, relative to PSI and cyt-bf, and PC, in concert with cyt-bf, is a key component that adjusts to control the electron transport rate. PC dependent flux control may serve to adjust the electron transport rate under conditions where the carbon assimilation is diminished and thereby protects PSI against over-reduction and reactive oxygen production.

Light- and temperature-induced changes in the distribution of excitation energy between Photosystem I and Photosystem II in spinach leaves

Abstract The influence of light quality and temperature on the distribution of the absorbed quanta between Photosystem I (PS I) and Photosystem II (PS II) in spinach leaves has been studied from the characteristics of chlorophyll fluorescence at 77 K. Leaves were preilluminated at different temperatures with either PS I light (to establish State 1) or with PS II light (to establish State 2), then cooled to 77 K and measured for fluorescence. In State 1, energy distribution appeared to be unaffected by temperature. A transition to State 2 resulted in an increase in PS I fluorescence and a decrease in the PS II fluorescence, indicating that a larger fraction of energy becomes redistributed to PS I. However, the extent of this redistribution varied: it was only small at 5°C to 20°C, but it largely increased at temperatures exceeding 20°C. This variation in the extent was related to a change in the mechanism of the state transition: at 15°C only the ‘initial’ distribution of energy was affected, while at 35°C an additional increase in the spill-over constant, kT (II → I), was included. It is assumed that under physiological conditions kT (II → I) is under the control of temperature rather than of light quality, whereby in leaves adapted to high physiological temperatures, the probability of energy spill-over from closed PS II centres to PS I is enhanced. In darkened leaves, the spill-over constant has been manipulated by preincubation at different temperatures. Then, the light-induced ‘energization’ of thylakoid membranes has been tested by measuring the light-induced electrochromic absorbance change at 515 nm (and light-induced light-scattering changes) in these leaves. The flash-induced 515 nm signal as well as the initial peak during a 1 s illumination were not affected by energy distribution. However, the amplitude of the pseudo-steady-state signal (as established during 1 s illumination) was considerably enhanced in leaves in which a larger fraction of the absorbed energy is distributed to PS I at the expense of PS II excitation. The results have been interpreted in such a way that an increase in energy spill-over from PS II to PS I favours a cyclic electron transport around PS I. It is discussed that changes in energy distribution (via spill-over) may serve to maintain a suitable balance between non-cyclic and cyclic electron transport in vivo.

Imaging of chlorophyll-a-fluorescence in leaves: Topography of photosynthetic oscillations in leaves of Glechoma hederacea

Images of chlorophyll-a-fluorescence oscillations were recorded using a camera-based fluorescence imaging system. Oscillations with frequencies around 1 per min were initiated by a transient decrease in light intensity during assimilation at an elevated CO2-concentration. The oscillation was inhomogenously distributed over the leaf. In cells adjacent to minor veins, frequency and damping rate was high, if there was any oscillation. In contrast, the amplitude was highest in cells most distant from phloem elements (maximal distance about 300 μm). The appearance of minor veins in oscillation images is explained by a gradient in the metabolic control in the mesophyll between minor veins and by transport of sugar from distant cells to phloem elements. The potential of fluorescence imaging to visualize ‘microscopic’ source-sink interactions and metabolic domains in the mesophyll is discussed.

Photosynthetic Control of Electron Transport in Leaves of Phaseolus Vulgaris: Evidence for Regulation of Photosystem 2 by the Proton Gradient

Under physiological conditions, the general distribution of control between different processes (eg. stomata, carbon metabolism, electron transport, photochemisty) may be complex and is always shared to some extent among elements (5). The primary consideration of the analysis reported here is the regulation of chloroplast membrane reactions when carbon metabolism clearly limits the rate of net photosynthesis. This condition will be referred to as ‘photosynthetic control.’ We show how the restriction of carbon metabolism feedsback, regulating the primary photochemisty of both photoreactions to match the rate at which their products can be accepted by the biochemical reactions. A new mechanism is proposed which relates the high-energy quenching of chlorophyll fluorescence (7) to regulation of the quantum yield of PS2 photochemistry.

Regulatory Subunit B′γ of Protein Phosphatase 2A Prevents Unnecessary Defense Reactions under Low Light in Arabidopsis

Light is an important environmental factor that modulates acclimation strategies and defense responses in plants. We explored the functional role of the regulatory subunit B′γ (B′γ) of protein phosphatase 2A (PP2A) in light-dependent stress responses of Arabidopsis (Arabidopsis thaliana). The predominant form of PP2A consists of catalytic subunit C, scaffold subunit A, and highly variable regulatory subunit B, which determines the substrate specificity of PP2A holoenzymes. Mutant leaves of knockdown pp2a-b′γ plants show disintegration of chloroplasts and premature yellowing conditionally under moderate light intensity. The cell-death phenotype is accompanied by the accumulation of hydrogen peroxide through a pathway that requires CONSTITUTIVE EXPRESSION OF PR GENES5 (CPR5). Moreover, the pp2a-b′γ cpr5 double mutant additionally displays growth suppression and malformed trichomes. Similar to cpr5, the pp2a-b′γ mutant shows constitutive activation of both salicylic acid- and jasmonic acid-dependent defense pathways. In contrast to cpr5, however, pp2a-b′γ leaves do not contain increased levels of salicylic acid or jasmonic acid. Rather, the constitutive defense response associates with hypomethylation of DNA and increased levels of methionine-salvage pathway components in pp2a-b′γ leaves. We suggest that the specific B′γ subunit of PP2A is functionally connected to CPR5 and operates in the basal repression of defense responses under low irradiance.

Photosynthetic control, “energy-dependent” quenching of chlorophyll fluorescence and photophosphorylation under influence of tertiary amines

The effects of the tertiary amines tetracaine, brucine and dibucaine on photophosphorylation and control of photosynthetic electron transport in isolated chloroplasts of Spinacia oleracea were investigated. Tertiary amines inhibited photophosphorylation while the related electron transport decreased to the rates, observed under non-phosphorylating conditions. Light induced quenching of 9-aminoacridine fluorescence and uptake of 14C-labelled methylamine in the thylakoid lumen declined in parallel with photophosphorylation, indicating a decline of the transthylakoid proton gradient. In the presence of ionophoric uncouplers such as nigericin, no effect of tertiary amines on electron transport was seen in a range of concentration where photophosphorylation was inhibited. Under the influence of the tertiary amines tested, pH-dependent feed-back control of photosystem II, as indicated by energy-dependent quenching of chlorophyll fluorescence, was unaffected or even increased in a range of concentration where 9-aminoacridine fluorescence quenching and photophosphorylation were inhibited. The data are discussed with respect to a possible involvement of localized proton flow pathways in energy coupling and feed-back control of electron transport.

Differential sensitivity to dibucaine of photosynthetic control of electron transport and photophosphorylation in chloroplasts

Abstract The effect of dibucaine on the photosynthetic control of electron transport, as related to acidification of the thylakoid lumen, and photophosphorylation in isolated chloroplasts of Spinacia oleracea was studied and compared to that of a typical uncoupler (nigericin) and energy transfer inhibitor (3′- O -napthyl-ADP). In some respects, dibucaine resembled an uncoupler: it inhibited photophosphorylation and decreased quenching of 9-aminoacridine fluorescence as well as uptake of [ 14 C]methylamine and inhibited the intrathylakoid accumulation of H + , as measured by a glass electrode. The preactivated thylakoid ATPase was stimulated by dibucaine, although this stimulatory effect was to some extent superimposed by an inhibition of the enzyme. However, as opposed to what is expected for an uncoupler, dibucaine did not release the pH-dependent control of electron transport. In addition, pH-dependent high-energy quenching of chlorophyll fluorescence, normally inhibited by uncouplers, was not affected by dibucaine. It is concluded that dibucaine selectively reduces the driving force of photophosphorylation, while pH-dependent control of electron transport remains largely unaffected. The data are discussed regarding current models on coupling of photophosphorylation and on regulation of electron transport.